Successful engineering of biomimetic skeletal muscle tissues could allow creation of accurate models of muscle physiology and disease and aid treatment of various muscle disorders. This project is based on our recently developed methods to utilize adult rat myogenic cells for engineering of 3D skeletal muscle tissues with structural and functional properties comparable to those of native muscle. Specifically, we have established conditions for robust in vitro expansion of adult rat myogenic cells and have successfully utilized them to engineer skeletal muscle tissues with contractile capacity 10- 100 fold higher than previously reported. Importantly, in a comprehensive set of preliminary studies we for the first time show that self-regenerative capacity of adult-derived engineered muscle in vitro and survival in vivo can be significantly enhanced by a 3-D co-culture of skeletal muscle progenitors with non-polarized macrophages derived from bone marrow. We propose to build on these exciting results and systematically explore the use of 3D muscle-macrophage co-culture system to create highly contractile and regenerative muscle tissues with the capacity for rapid vascular and neuronal integration and successful repair of skeletal muscle injury in vivo. We will study: (1) the cellular and molecular mechanisms of macrophage mediated self-repair of tissue-engineered muscle in vitro, (2) the combined effects of macrophages, vascular cells, and biophysical cues on the ability of in vitro formed pre- vascularized engineered muscle to undergo rapid blood perfusion and functional maturation in vivo, and (3) the roles of macrophage supplementation and synaptogenic stimulation in vitro upon the ability of muscle-macrophage implants to functionally integrate with and repair damaged skeletal muscle in vivo. Successful completion of the proposed studies will establish foundation for the future applications of tissue engineering methodologies to human muscle repair. Furthermore, our novel strategy to utilize immune system cells as pro-regenerative adjuvants inside tissue-engineered implants may find broad applications in the field of regenerative medicine.